Use of secondary cell wall polymer of procaryotic microorganisms

ABSTRACT

A compound body can be constructed, using secondary cell wall polymers of prokaryotic microorganisms as bonding agent to anchor a monomolecular lattice to a support, with or without functional groups attached to the lattice. The lattice may be comprised of lipid or crystalline cell surface layer proteins. The support is illustrated by a micro-filtration membrane, a cell surface ultra-filtration membrane (SUM), and a micelle, respectively.

The invention refers to the use of the secondary cell-wall polymer ofprokaryotic microorganisms and to compound bodies composed of onecarrier and one molecular layer.

Crystalline cell surface layers (the so-called S-layers, “S” forsurface) are some of the most common outermost cell envelope componentsof prokaryotic organisms (archaebacteria and eubacteria). S-layers aremade up of single protein or glycoprotein species (Mr approx. 50,000 to2000,000). [Sleytr, U. B., P. Messner, D. Pum and M. Sára. (eds) 1996.Crystalline Bacterial Cell Surface Proteins. In: Molecular BiologyIntelligence Unit. Academic Press. R. G. Landes Company. Austin, USA;Sleytr, U. B., P. Messner, D. Pum and M. Sára. 1999. CrystallineBacterial Cell Surface Layers (S-layers): from Supramolecular CellStructure to Biomimetics and Nanotechnology. Agnew. Chem. Int. Ed.38:1034–1054; Sleytr, U. B. and T. J. Beveridge. 1999. BacterialS-layers. Trends Mocrobiol. 7(6):253–260.] Isolated S-layer(glyco)proteins of many organisms have the capacity for self-assemblyinto monomolecular crystalline lattices in suspension, on solid supports(for example, silicon wafers, polymers, metals), on the air-waterinterface as well as spread-out lipid films and liposomes. S-layers showpores of regular size and morphology. Permeability studies have shownthat S-layers demonstrate sharp molecular mass separation limits in theultra-filtration area. These properties have also led to the use ofS-layers in the manufacture of ultra-filtration membranes (specificationof the European patent No. 0 154 620 B1).

Comprehensive pilot tests have shown that S-layer (glyco)protein ofnumerous Bacillaceae can recrystallize on spread-out lipid films (forexample, phospholipids, tetraetherlipids and other amphiphilicmolecules). For this to happen, the lipids are first spread on theair-water interface in a trough according to the Langmuir-Blodgetttechnique to form a monolayer. It can be advantageous for therecrystallization of S-layer (glyco)proteins on the lipid film that thespread-out lipid film is placed between barriers at a defined spreadingpressure. To recrystallize the S-layer (glyco)proteins on the spread-outlipid film, the dissolved S-layer (glyco)proteins are inserted into thesubphase where they reach the spread-out lipid film either by diffusionor active mixture of the subphase. The pH-value, ionic strength andionic composition of the subphase can then influence therecrystallization behavior of the S-layer (glyco)proteins. [Sleytr, U.B., P. Messner, D. Pum and M. Sára. (eds) 1996. Crystalline BacterialCell Surface Proteins. In: Molecular Biology Intelligence Unit. AcademicPress. R. G. Landes Company. Austin, USA; Sleytr, U. B., P. Messner, D.Pum and M. Sára. 1999. Crystalline Bacterial Cell Surface Layers(S-layers): from Supramolecular Cell Structure to Biomimetics andNanotechnology. Agnew. Chem. Int. Ed. 38:1034–1054; Sleytr, U. B. and T.J. Beveridge. 1999. Bacterial S-layers. Trends Mocrobiol. 7(6):253–260.]

The recrystallization in the form of a closed monomolecular S-layercomes from nucleation seeds (crystallites). The singular crystallineS-layer domains grow until they meet each other and unite into acontiguous layer. During the recrystallization of the S-layers, bilayerscould also form depending on the selected conditions. In these bilayers,the single layers can bind with their inner or outer sides. Pilot testshave shown that S-layer-supported lipid films demonstrate significantlyhigher mechanical stability as “naked” lipid films. Further pilot testshave shown that the recrystallization of an S-layer does not affect thefunctionality of lipid membranes. During the build-up of a contiguousassociated S-layer, the specific conductivity and specific capacitanceof lipid membranes do not change significantly. Sleytr, U. B., P.Messner, D. Pum and M. Sára. 1999. Crystalline Bacterial Cell SurfaceLayers (S-layers): from Supramolecular Cell Structure to Biomimetics andNanotechnology. Agnew. Chem. Int. Ed. 38:1034–1054. [Schuster, B.; U. B.Sleytr, A. Diederich, G. Bähr and M. Winterhalter. 1999. Probing thestability of S-layer-supported planar lipid membranes. Eur. Biophys. J.28:583–590; Pum, D. and U. B. Sleytr. 1999. The application of bacterialS-layers in molecular nanotechnology. Trends Biotechnol. 17:8–12].Functional molecules can build up before and after the recrystallizationof S-layers in the lipid membranes. These functionality studies weredone using voltage clamps and black-lipid membrane technology.[Schuster, B., D. Pum and U. B. Sleytr. 1998. Voltage clamp studies onS-layer-supported tetraether lipid membranes. Biochim. Biophys. Acta1369: 51–60; Schuster, B., D. Pum, H. Bayley and U. B. Sleytr. 1998.Self-assembled α-hemolysin pores in an S-layer-supported lipid bilayer.Biochim. Biophys. Acta 1370: 280–288]. Using the example of α-hemolysin(α-HL), it could be shown that heptamer pores (comprising 7 identicaltransmembrane pores made of α-HL molecules) build up only when thepore-forming molecules are raised from the lipid side. Because of themolecular filtering action of the S-layer lying in the ultra-filtrationarea, however, the α-HL molecules do not penetrate the S-layer latticeto reach the lipid membrane. On the other hand, it was shown thatsmaller molecules (e.g., ionic channels such as valinomycin) are raisedfrom both sides, integrate into the lipid membrane and, as aconsequence, can be measured as functional ionic channels usingelectrophysiological methods. The interaction between monomolecular andbimolecular lipid films (including mixtures with other amphiphilicmolecules such as hexadecylamine) and liposomes with S-layer(glyco)protein lattices has so far been investigated using a widespectrum of biophysical methods. Hirn, R., B. Schuster, U. B. Sleytr andT. M. Bayerl. 1999. The effect of S-layer protein adsorption andcrystallization on the collective motion of a planar lipid bilayerstudied by dynamic light scattering. Biophys. J. 77:2066–2074.[Schuster, B.; U. B. Sleytr, A. Diederich, G. Bähr and M. Winterhalter.1999. Probing the stability of S-layer-supported planar lipid membranes.Eur. Biophys. J. 28:583–590; Hianik, T., S. Küpcü, U. B. Sleytr, P.Rybár, R. Krivánek and U. Kaatze. 1999. Interaction of crystallinebacterial cell surface proteins with lipid bilayers in liposomes. Asound velocity study. Colloids Surfaces A 147: 331–339; Mader, C., S.Küpcü, M. Sára and U. B. Sleytr. 1999. Stabilizing effect of an S-layeron liposomes towards thermal or mechanical stress. Biochim. Biophys.Acta 1418: 106–116; Györvary, E., B. Wetzer, U. B. Sleytr, A. Sinner, A.Offenhäuser and W. Knoll. 1999. Lateral diffusion of lipids in silane-,dextran- and S-layer-supported mono- and bilayers. Langmuir15:1337–1347: Weygang, M., B. Wetzer, D. Pum, U. B. Sleytr, N.Cuvillier, K. Kjaer, P. B. Howes and M. Lösche. 1999. Bacterial S-layerprotein coupling to lipids. X-ray reflectivity and grazing incidencediffraction studies. Biophys. J. 76: 458–468; Wetzer, B., A. Pfandler,E. Györvary, D. Pum, M. Lösche and U. B. Sleytr. 1998. S-layerreconstitution at phospholipid monomoleculars. Langmuir. 14: 6899–6909].It was shown that the recrystallization of an S-layer lattice can have asignificant effect on the properties of the lipid film (for example,fluidity) and molecular layout. [Györvary, E., B. Wetzer, U. B. Sleytr,A. Sinner, A. Offenhäuser and W. Knoll. 1999. Lateral diffusion oflipids in silane-, dextran- and S-layer-supported monolayers andbilayers. Langmuir. 15:1337–1347]. Basically, S-layer-supported lipidmembranes correspond to the supramolecular building principle of thecell envelope of those archaebacteria (Archaee) that have only oneS-layer as a cell wall component apart from the cystoplasmic membrane.But since isolated S-layer (glyco)proteins of archaebacteria aresignificantly more difficult to recrystallize on lipid films, theS-layer glyco(proteins) of other prokaryotic organisms (for example,Bacillaceae) are preferable. The manufactured composite S-layer lipidfilms are then biomimetic structures that are adapted to the cellenvelopes of Archaebacteria, etc. and the native components of theArchaebacteria need not be used.

From work and publications of the applicants it is known that theS-layer (glyco-protein) of Gram-positive bacteria can be bonded to theunderlying rigid cellular wall layer (the so-calledpeptidoglycan-containing layer) with very specific reciprocal actiontaking place. This specific bonding evidently occurs often between theS-layer protein and the so-called secondary cell wall polymers (referredto below in the text as “SCWP”). These polymers are bonded covalently tothe matrix of the peptidoglycan layer and can be separated from it (forexample, by treating it with HF) and extracted in pure form. From thecomparison of amino acid sequences of various S-layer proteins, domainswere traced that are responsible for the bonding of the S-layer to thepeptidoglycan-containing cellular wall layer, especially the SCWPcomponents.

As is already known from Pum, D. and U. B. Sleytr. 1999. The applicationof bacterial S-layers in molecular nanotechnology. Trends Biotechnol.17:8–12 and Sleytr, U. B. and M. Sára. 1997. Bacterial and archaealS-layer proteins: structure-function relationships and theirbiotechnological applications. Trends Biotchnol. 15:20–26, the S-layerhas a stabilizing effect on lipid membranes. These publications describeprocedures where the S-layer proteins in troughs were recrystallizedfrom the subphase on spread-out lipid films or on black lipid membranes.

The principle of SUM-supported lipid membranes [SUM: S-layerultra-filtration membrane] could also lead completely to new applicationpossibilities for functional lipid membranes. It is considerably simplerto bring a lipid film onto an SUM (for example, by means of theLangmuir-Blodgett or Langmuir-Schäfer method) or to assemble it directlyon the film than first to manufacture the lipid film by spreading it ona trough and then to recrystallize the S-layer proteins from thesubphase on the lipid film. The use of SUMs would also facilitate thedevelopment of new testing methods according to the principle of thevoltage-clamp measuring technique as they are required, for instance, inthe screening of pharmaceutical substances for their effect on themembrane-integrated or membrane-associated molecules (for example, ionicchannels, signal-transmitting molecules).

Moreoever, the voltage-clamp technique is also known: a delicatelyextracted pipette filled with buffer solution is carefully brought closeto a (functional) lipid membrane so that measurable contact ensues.Consequently, the trans-membrane functions are determined byelectrophysiological means (for example, by measuring the trans-membranecurrents). This widely established method, however, cannot be used forparallel studies as such studies are needed for high-throughputscreening (testing of many substances in one operational step).

At the present state of science, there are methods for manufacturingcomposite S-layer lipid films where the S-layer (glyco)protein arerecrystallized from a solution on lipid films (for example, Langmuirfilms, black lipid membranes) in mesoscopic and macroscopic dimensions.

Among other things, the object of the invention is the task to find newways to stabilize lipid films.

The invention resolves this task by using secondary cell wall polymersfor the directed monomolecular bonding of (functional) molecules,preferably molecular layers and/or for the attachment of functionalmolecules on molecules of a support. In this manner, the specificinteractions between one or more domains of the S-layer proteins and theso-called secondary cell wall polymers are used for makingsupra-molecular structures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the use of S-layer ultra-filtration membranes.

FIG. 2 shows the chemical coupling of the SCWP to another polymer.

FIG. 3 shows S-layers on the polymer surface functioning with the SCWP.

FIG. 4 shows the use of S-layer fusion proteins that are differentlyfunctionalized.

FIG. 5 shows the use of amphiphilic molecules consisting of SCWP andhydrophobic chains.

FIG. 6 show the use of molecules with one central hydrophobic part andtwo hydrophilic domains.

FIG. 7 shows composite SUM/lipid membranes.

FIG. 8 shows the making of composite S-layer/functional lipid membranesusing SCWPs.

FIG. 9 shows the use of the SCWP system to accelerate vesicle fusion.

FIG. 10 shows that SU should be mounted at the end of a glass or plastictube to make a SUM-spanning aperture.

FIG. 11 shows SUM tubes, separately or together.

FIG. 12 shows micelles with S-layer proteins.

The making of these S-layer membranes is already the subject of a patentissued in many states (see, for instance, specifications of Europeanpatent 0154620 B1). Basically, this procedure involves the attachment ofS-layer fragments or cell wall fragments of S-layer-supportingProkaryotes onto porous supports; (preferably) micro-filtrationmembranes with open-cell, spongiform structure or membranes that weremade according to the radiation-damage track technology (for example,nucleopore membranes). After the attachment of a contiguous S-layerposition (this can also consist of several individual layers), theS-layer lattices are chemically cross-linked intra and inter-molecularly(preferably with glutaraldehyde). When using glutaraldehyde as achemical cross-linker, it is preferable to reduce the ensuing Schiff'sbases with boron hydride to increase the chemical stability of theS-layer membranes. The S-layer membranes made this way are used asultra-filtration membranes because of the molecular filtering action ofthe S-layer lattice. These S-layer ultra-filtration membranes willhenceforth be abbreviated to SUMs.

By using SUMS as support for functional lipid membranes (or membranesthat consist of other film-building, say, amphiphile, molecules),significant technical and application-related advantages can be achievesin comparison to the aforementioned procedure for recrystallizingS-layers on spread-out lipid films.

The attachment or the creation of (functional) lipid films on SUMS canbe done in many various ways:

1. Immersion and/or emersion of the SUMS from a lipid film spread out ona trough (for example, Langmuir-Blodgett or Langmuir-Schäfer technique).While a “functional” lipid membrane can be made by simple immersion oremersion using a spread-out tetraether lipid film, two steps (immersionand emersion sequences) are necessary for phospholipids, for instance.

2. Manufacturing a chemically bonded lipid monolayer on the S-layerlattice where the hydrophobic parts of the amphiphilic molecules stayaway from the SUM. The second lipid monofilm is then attachedaccordingly using the Langmuir-Blodgett or Langmuir-Schäfer technique(see above).

3. Certain functions (for instance, biotinylation, cysteine residues,streptavidine bonding domains) can be built into the S-layer lattices.In this way, the corresponding SUM surfaces can deal with a specificinteraction with lipid molecules or other amphiphilic molecules. This ishow composite S-layer/lipid membranes are made where the lipid film isbonded to the S-layer (glyco)protein matrix of the SUM according to therepetitively occurring specific bonding position.

4. After the lipid film is attached to the SUM, a covalent chemicalcross-linking of head groups of lipid films (or the amphiphilicmolecules) occurs with the S-layer matrix. This cross-linking reactionpreferably happens after the cross-linking substance permeates throughthe porous support of the SUM (for instance, through themicro-filtration membranes).

It is advantageous that the secondary cell wall polymers can bindthrough the respective domains of the functional molecules, preferablythe molecular layers and/or molecules of the support. One the one hand,this allows one to pinpoint exactly the functional molecules ormolecular layers and, on the other, align them precisely. The secondarycell wall polymers can then bind to the molecules, preferably themolecular layers or molecules of the support, through lectin-like bonds,which also facilitates an exact selection of the domains or epitopes forbonding the molecules or molecular layers to the supports.

A preferred application in the invention involves having the secondarycell wall polymers as the bonding agents between the polymer supportstructures and the function molecules, molecular groups or molecularlayers, where the polymer support structures can be loaded directly withthe appropriate functional molecules. The support structures can then bemicro-filtration membranes. In addition, the molecular layers can comein the form of monomolecular crystalline protein layers, where, on theone hand, appropriate filters with sharp cut-off can be made because ofthe crystalline structure of the protein layer and, on the other, theseprotein molecules, in turn, can be loaded directly or indirectly withfunctional molecules. The monomolecular crystalline protein layers, inturn, act as a support for a functional lipid membrane, achieving verystable composite structures that, for example, can be used forhyperfiltration and the like. Furthermore, one or more identical ordifferent functional domains can brought onto that side of themonomolecular crystalline protein layers averted from their secondarycell wall polymers, with the protein layers used as mediator for anarranged bonding of other molecules. To achieve special bondingpossibilities, the monomolecular molecules can have amphiphilecharacteristics and consists of hydrophobe chains and hydrophilesecondary cell wall polymers. This allows one to control the directionof the attachment to a aqueous or non-aqueous phase. Furthermore, themolecules of the support can be monomolecular crystalline protein layerswhere the secondary cell wall polymers make up the bonding agents to afunctional lipid layer during the formation of an aggregate compoundbody. The lipid molecules are distanced accordingly from the proteinlayers in such a way that the lipid layer is firmly bonded to theprotein layers. So through functional lipid layers two aggregatecompound bodies can then be bonded as mirror images of each other. Thisallows the build-up of layers according to the so-called sandwichprinciple. The monomolecular crystalline protein layers can consist ofidentical or different protein molecules, which make it possible forvarious bonding domains, charges, functional molecules and the like tobe present on the outer layer of the compound body, thus greatlyexpanding the scope of application.

Finally, the functional lipid layer may have a vesicular structurewhich, according to its nature, allows artificially adjusted compoundbodies.

As already mentioned, the invention also refers to a compound bodyconsisting of a support and a molecular layer which is thus marked inthe invention that the molecules of this molecular layer are bonded tothe support, in a monomolecular direction, through secondary cell wallpolymer chains according to the invention's application. Thisfacilitates the build-up of a precisely structured, precisely definedcompound body chemically and functionally.

The secondary cell wall polymer chains can anchor on the support and/orthe bonding molecules through a lectin-like bond, where one of thepassing domains of the protein molecule attains an appropriate bond. Toobtain a stable compound body, monomolecular crystalline protein layerscan be bonded on a polymer matrix through the secondary cell wallpolymers where these secondary cell wall polymers act as the anchor andthe alignment and/or the target distribution. Lipid films or vesiclescan also be configured on that side of the monomolecular crystallineprotein layers averted from the secondary cell wall polymers, providingadditional application possibilities. Furthermore, functional domainscan be designated in regular intervals and configurations on that sideof the monomolecular crystalline protein layers averted from thesecondary cell wall polymers, allowing a targeted attachment of otherfunctional molecules on the protein layers.

To achieve different kinds of bonds or distribution of functionalmolecules, the functional domains can be provided in different butregular patterns.

Moreover, the secondary cell wall polymers of one monomolecularcrystalline protein layer, which preferably is attached directly onto asupport, jut out and bond to a functional lipid layer. This produces astable support layer because of the protein layer directly attached tothe support: the lipid layers are bonded to the secondary cell wallpolymers free from interactions of the protein layer. Two such proteinlayers can then bind through the functional lipid layer opposite eachother as mirror images, leading to a stable sandwiched compound body.The one protein layer can consist of other protein molecules as theother protein layer so that different bonding relationships, domains andfunctions can exist on both sides on the compound body.

As a variant of the known voltage-clamp technique described above, thecompound body can be configured so that it spans the opening at one endof a small tube. In comparison to the known open pipette, such a smalltube equipped with the compound body used in the invention (referred tohereinafter as the SUM tube) can be brought near the spread-outfunctional lipid membrane much more quickly without disturbing the film.Numerous SUM tubes laid out beside each other can then be usedsimultaneously, for example, for measurements conducted on functionallipid membranes. Basically, one can conceive of measuring procedureswhere the first step involves layering all SUM tubes with functionallipid membranes. This could be done by immersing them in a spread-outtetraether lipid film in which, in a preceding step, functionalmolecules are built in. [Schuster, B., D. Pum and U. B. Sleytr. 1998.Voltage clamp studies on S-layer-supported tetraether lipid membranes.Biochim. Biophys. Acta 1369:5–60]. In the second step, the SUM tubeswould be lowered into small vessels (for instance, similar microtiterplates) containing various substances whose effect on themembrane-associated or membrane-integrated functional molecules shouldbe tested. The electrophysiological measurements would then be doneaccording to the classic voltage-clamp technique.

Together with SCWP, SUMS could also serve as support for functionallipid membranes in the macroscopic area. Currently, there are hardly anycomparable support layers for the large-surface stabilization of lipidmembranes.

Finally, the compound body used in the invention can be used as supportfor special functional molecules for diagnostics, drug targeting, drugdelivery, artificial virus drug screening, high-throughput screening orthe like.

With the use described in the invention, vesicular structures can alsobe created: for example, liposomes or lipid drips (lipid particles)coated with S-layers.

Finally, a micelle composed of hydrophobic self-assembling chains canalso be used as support.

The diagram shows an enlarged cross-section of various configurations ofthe compound body.

FIG. 1 shows the basic use of S-layer ultra-filtration membranes withthe following abbreviations used in all pictures:

-   -   MF: micro-filtration membrane,    -   S-L: S-layer,    -   FL: functional lipid membrane, that is, lipid membrane with        built-in membrane function, especially trans-membrane function        (e.g., ionic channel).

Such a configuration is already the state of the art of the applicant'sown work.

FIG. 2 shows the chemical coupling of the SCWP to another polymer andthe resulting mixture of these molecules with a polymer usually used formaking micro-filtration membranes (with spongiform structure). Duringthe illuviation process in aqueous solutions (e.g., according to thephase inversion procedure), hydrophilic SCWPs should jut out into theaqueous phase while the polymer chains (covalently) bonded to them areanchored in the compact polymer structure.

Basically, one should also think of other possibilities to functionalizethe surfaces of polymer structures (films, spongiform or other porousstructures) with SCWPs. In all of these procedures the hydrophilic SCWPchains jut out at a sufficient distance from the surface of the polymerstructures to allow contact to the corresponding bonding domains of theS-layer proteins.

Besides the polymerization procedure, chemical coupling methods can alsobe used. For example, reactive groups could be built into one end of theSCWP. These groups can then react with groups on the surface of thepolymer structures. Conversely, pre-activated groups can be brought toreact with reactive groups on the SCWP.

As shown in FIG. 3, S-layers on the polymer surface functioning with theSCWP can then be recrystallized from a solution of S-layer(glyco)protein. This method would lead to a complete monomolecularS-layer coating of the polymer surface. Since S-layer fusion proteinswith built-in specific functions (for example, biotin binding domains ofthe streptavidin, protein A, protein G, antibody or antigen domains) canbe used for the recrystallization besides native S-layer(glyco)proteins, a very uniform functionalization of polymer matricescan be achieved using this method (see above).

With the use of S-layer fusion proteins according to FIG. 4, two or moreidentical but differently functionalized S-layer proteins could also beused simultaneously. In this manner, a uniform S-layer lattice wouldbuild up on the SCWP because of the uniform protein base structure. Thislattice, however, would have different bonding positions (functionaldomains) in discrete intervals (but statistically distributed).

FIG. 5 shows the use of amphiphilic molecules consisting of SCWP andhydrophobic chains that correspond, for instance, to glycolipids intheir molecular structural concept. Similar molecules are also found,say, in the plasma membranes of Archaebacteria (Archaea). The diagram,in particular, shows the making of glycolipid-equivalent molecules.

FIG. 6 shows the use of naturally occurring or fully synthetic moleculesconsisting of one central hydrophobic part and two hydrophilic domains.

FIG. 7 illustrates the use of the molecules shown in FIG. 5 and FIG. 6for making composite SUM/lipid membranes. The use of the amphiphilicmolecules allows wide variation in configuring composite SUM/lipidmembranes. The basic possibility to use two different SCWPs that bind todifferent S-layer proteins deserves special mention.

FIG. 8 illustrates the making of composite S-layer/functional lipidmembranes by using SCWPs. The following abbreviations are used in FIG.8:

-   -   A: S-layer protein type A    -   SCWP-A: secondary cell wall polymer that binds specifically to        type A S-layer proteins,    -   B: type B S-layer protein    -   SCWP-B: secondary cell wall polymer that binds specifically to        type B S-layer protein.

An S-layer (for example, type A) is first brought on themicro-filtration membrane. Basically, this process corresponds to theabove-mentioned procedure for making SUMs. Consequently, a monolayer ofamphiphilic molecules (suited for making functional lipid membranes, seeexamples) is brought on the SUM by using either the Langmuir-Schäfertechnique or the Langmuir-Blodgett technique. The monolayer is connectedto the SUM by means of the specific interaction of the SCWP part of theamphiphilic molecules with the S-layer with the SUM. This anchoring oflipid monolayers onto S-layers on SUMs led to a significant increase instability and longevity of functional lipid membranes.

As an alternative to the molecules described in FIG. 5, the moleculesspecified in FIG. 6 can also be used to make functional lipid membraneswhere the latter facilitate the making of a functional lipid membrane inone step. By using the molecules shown in FIG. 5, on the contraty,another monolayer of amphiphilic molecules can be applied in a secondstep. If this outer layer contains amphiphilic molecules with a SCWPpart, the exposed SCWP chains can serve as specific bonding positionsfor another S-layer. This second S-layer can, for example, berecrystallized on the lipid membrane from a solution of S-layer(glyco)proteins. Alternatively, the S-layer can also be recrystallizedin the first step on the air-water interface and only then applied tothe lipid membrane (say, by immersion).

The supra-molecular layer system shown in FIG. 8 can be done by usinguniform or different SCWP-S-layer partners. The diagram shows examplesof type A S-layer and type A SCWP as well as type A S-layer and type ASCWP with type B S-layer and type B SCWP.

To attach lipid membranes on supports, the so-called liposome fusiontechnique is used multiple times. Under this method, the lipid membraneis offered as a lipid vesicle (so-called liposomes). Preferably thedesired funcational molecules are already built into the liposomes.Under appropriate trial conditions, the lipid vesicles open upon contactwith the surface of the solid support to finally form a contiguous lipidfilm.

As shown in FIG. 9, the SCWP system can also be used, where necessary,to accelerate this vesicle fusion and secure the bonding of the lipidfilm to the SUM.

Using SUM-supported (stabilized) functional lipid membranes forhigh-throughput screening.

There are basically two measuring setups:

-   -   a) voltage clamp or patch clamp methods    -   b) black lipid membrane methods

In both cases, the use of SUMs should extend the lifetime and thus theusability of functional lipid membranes.

Making SUM Tubes

As shown in FIG. 10, the SUM should be mounted at the end of a glass orplastic tube resulting in an SUM-spanning aperture. In principle, thispart would correspond to the opening of a patch clamp or voltage clamppipette, with the difference that the (functional) lipid membrane doesnot span over the pipette opening but is supported by the SUM. Mountingthe lipid membrane and its functioning could follow the methods alreadydescribed above.

Another advantage in using SUM tubes is that after applying the lipidmembrane another S-layer could be applied by deposition orrecrystallization (see FIG. 7 and FIG. 8). This additional S-layer wouldfulfill a (further) stabilizing and protective function. In thiscontext, one has to recall the sharp molecular cut-off of the S-layers.

After the lipid membranes are applied to the SUM, or before or after thepossible recrystallization of another S-layer, an intermolecular and/orintramolecular chemical cross-linking of the S-layer (glycoprotein)matrix and of the lipid film can occur. For this to happen, appropriatecross-linkers must be applied to the aqueous phase.

The SUM tubes can be used either separately or in groups (see FIG. 1). Agroup configuration allows the application of the lipid membranes in oneoperating step and, consequently, the performance of parallelmeasurements in any number of vessels containing various substanceswhose effect on the functionalized membranes should be tested. Whenusing SUM tubes, active substances could also be added directly to theglass tubes filled with buffer solutions or water. Furthermore, wherenecessary, the environmental conditions (e.g., ionic strength, ioniccomposition, pH-value, temperature) in the tubes and, separately, in thevessels where they are immersed could also be altered without possiblepressure fluctuations affecting the integrity of the functional lipidfilm. This same increased stability of the SUM-associated lipidmembranes mentioned in the outset as compared to the pressure changesmerits special mention in applications for the invention. The advantagesmentioned above can also be applied directly to the test setups of blacklipid membranes.

In conjugates consisting of one SCWP-(carbohydrate)part and a functionalmolecular part, the carbohydrate part can consist of an intact SCWP,partial structures (oligosaccharids fragments) made from SCWP bychemical breakdown or synthetically manufactured partial structures ofthe SCWP (oligosaccharide derivatives).

The functional molecular part can have the function of being a bond tolipid phases (for example, liposomes) or gold, a secure anchor to asolid phase (for example, S-layer), polymerization capacity (theconstructed conjugate acts as a co-monomer), pharmaceutical effect,combined function such as bonding to lipid phases and immuno-stimulatedeffect (lipid-A-derivative, type FK-565 lipopeptide) or, finally,detectability (e.g., fluorescence).

Chemically, the linking of the carbohydrate part with the functionalpart can occur in various ways, namely:

Reductive amination (formation of Schiff's bases with subsequentreduction to secondary amines).

-   -   Thiocarbamide formation or    -   Amide formation.

The reductive amination can occur on the carbonyl components of thecarbohydrate part through the dissociation of the reduced phosphoricacid residue from the SCWP with the formation of reduced SCWPs, throughpartial De-N-acetylization and desaminization with nitrous acids withthe formation of anhydromannose (anhydroglucose?) end groups, throughβ-elimination of SCWP containing uronic acid, or through breakdown ofperiodate (possible partial breakdown). The reductive amination can becarried out on the functional part of the amino components by usingehtylenediamine, propolenediamine or by introducing an amino group onthe reduced end of the SCWP by using allylamine, aminomethacrylic acidand its derivatives, other amino compounds with unsaturated groups(Aldrich catalog) or S-layers (ε-lysin-NH₂ groups). A carbonyl componentreacting with ethylenediamine or another diamine compound can result inamino groups on the carbohydrate part. The carbonyl components on thefunctional part can acquire a Smith product of S-layers orpolysaccharides.

The thiocarbamide can form through FITC (fluorescein isothiocyanate) orthrough the conversion of primary amines in isothiocyanate throughreaction with thiophosgenes.

The amine formation can occur on the carbonic acid part of thecarbohydrate (SCWP) through lactal-lactone bromoxidation, on the aminocomponents similar to reductive amination, on the amino components ofthe carbohydrate (SCWP) through the formation and reaction of a carbonylcomponent with ethelenediamine or another diamine compound, and on thecarbonic acid part of the functional molecule as activator orlong-chained fatty acids and branched fatty acids, as activator ofasparagus acid or lipoic acid as well as activator of methacrylic acid(derivatives) and other unsaturated carbonic acids.

EXAMPLES

Group 1: Modification of Secondary Cell Wall Polymers

The secondary cell wall polymers (SCWP) were extracted frompeptidoglycan-containing Sacculi of various Bacillaceae by using 48%hydrogen fluoride (HF) based on the specifications of von Ries, et al(1997), cleaned by gel filtration chromatography, the eluates dialyzedagainst A. purify., the inner dialysate frozen at −20° C. andlyophilisated. The organisms used whose S-layer proteins and SCWP havethe following name: Bacillus stearothermophilus PV72/p6 (Sára et al,1996), SbsA (Kuen et al, 1994), type A SCWP (Egelseer et al, 1998);Bacillus stearothermophilus ATCC 12980 (Egelseer et al, 1996), SbsC(jarosch et al, 2000), type A SCWP 9Egelseer et all, 1998); B.stearothermophilus PV72/p2 (Sára et al, 1996), SBsB (Kuen et al, 1997),type B SCWP (Ries et al, 1997; Sára et al, 1998); B. spaericus CCM 2177(Sára et al, 1989), SbpA, type C SCWP (Ilk et al, 1999);Thermoanaerobacter thermohydrosulfuricus L111-69 (Sára et al, 1988);Stta, type D SCWP. An exact listing of the properties of the individualS-layers and the corresponding SCWP can be found in Table 1.

TABLE 1 1. S-layer protein 2. Lattice type Genbank Interaction of 3.Overall length accession Main components of the SCWP/ the SCWP Organism4. Signal peptide number SCWP maximum MG with the Bacillus SbsA X 71092Type A Glucose:N-acetyl- N-terminus stearothermophilus hexagonalglucosamine:2,3-dideoxy-2,3- (AS 31-257 PV72/p6 1 228diacetamidomannuronic no SLH motifs) 30 acid = 1:1:2; structureclarified (MG_(max) 50,000) Bacillus SbsC AF055578 Type A same as aboveN-terminus stearothermophilus oblique (AS 31-257 ATCC 12980 1 099 no SLHmotifs) 30 B. stearothermophilus SbaD AF228338 Type A same as aboveN-terminus Mut 1 oblique (AS 31-257 940 no SLH motifs) 30 Bacillus SbsBX 98095 Type B N-acetyl- N-terminus stearothermophilus obliqueglucosamine:N-acetyl- (3 SLH motifs; PV72/p2 920 mannosamine = 2:1; AS33-204) 31 negatively loaded by pyruvate ketal, glycerine and uroic acidavailable; very complex configuration; structure not yet completelyclarified (MG_(max) 24,000) B. spaericus CCM SbpA AF211170 Type CN-acetyl- N-terminus 2177 cube glucosamine:N-acetyl- (3 SLH motifs; 1322 mannosamine = 2:1; AS 33-202) 30 each 2^(nd) N-acetylmannosamine hasa pyruvate ketal; structure clarified (MG_(max) 10,000) Th. SttA noneType D N-acetyl- N-terminus thermohydrosulfuricus hexagonalglucosamine:N-acetyl- L111-69 unknown mannosamine:Mannose = 1:0.5:1;loaded negatively with pyruvate ketal; structure not yet clarified(MG_(max) ~25,000)

Example 1

Conversion of Latent Aldehyde Groups (Reduced Ends) of the PolymerChains in Free Amino Groups

To modify the reduced ends (latent aldehyde groups) of the type A, B, Cand D SCWP, the following steps were performed: 10 mg of lyophilisatedmaterial were dissolved in 2 ml of saturated carbazole/hydrazidesolution (pH 6.2). Then 200 μl of sodium cyanoborohydride (NaBH3CN; 20mg/ml A. purify.) were added, 15 minutes at 100° C., then incubated for16 h at 90° C. To remove surplus reagents, it was dialyzed for 24 hagainst A. purif., the samples frozen at −20° C. and lyophilisated. Todetermine the free amino groups, 1 mg of the respective SCWP wasdissolved in 2 ml of 50 mM sodium hydrogen carbonate buffer (pH 7.8),and 100 μl of a solution butyloxycarbonyl-L-leucine-N-hydroxysuccinimideester (BOC Leuse; 3.5 mg/ml 100% ethanol) added. The reaction batch wasstirred for 18 h at 20° C., then for 6 h against a mixture of ethanol—A.purif., (30:7/v:v) and then dialyzed against A. purif. The innerdialysate was then lyophilisated and 0.5 mg of the lyophilisatedmaterial hydrolyzed with 6 N HCl for 6 h at 110° C. for the amino acidand amino sugar analysis (BIOTRONIK amino acid analyzer; Maintal, D.).The degree of modification of the polymer chains can be determined fromthe ratio of the glocosamine to the leucine. For all SCWPs, this wasfound to be >95%.

Example 2

Conversion of the Amino Groups Introduced in the SCWP into Thiole Groups

To convert the amino acids introduced in the SCWP, 5 mg of the SCWPmodified according to Example 1 was dissolved in 5 ml of 0.25 Mtriethanolamine-HCl buffer (pH 8.5). After adding 4 mg of2-iminothiolane (4-mercaptobutyrimidate), the solution was incubated for2 h at 37° C. under a nitrogen atmosphere, and finally dialyzed for 18 hat 4° C. against degassed A. purif. to remove the reagents. The innerdialysate was then frozen and lyophilisated. The number of thiole groupsintroduced was determined according to the Ellman method (1959). It wasshown that the amino groups could be converted quantitatively intothiole groups.

Example 3

Modification of Introduced Amino Groups Using Blotin

To modify the free amino groups (see Example 1), 5 mg of type A, B, C orD lyophilisated SCWP were dissolved in 20 mM of potassium-phosphatebuffer (pH 7.8) and 0.5 mg of sulfo-NHS-biotin (sigma) added. Themodification happened following the instruction recommended by thecompany Pierce. To remove surplus reagents, it was dialyzed for 18 h at4° C. against A. purif., the inner dialysate frozen at −20° C. andlyophilisated.

Example 4

Activation of the Freely Introduced Amino Groups

To activate the introduced amino groups, 5 mg of the lyophilisate fromExample 1 was dissolved in 500 μl of of p-phenylenediamine (10 mg/ml A.purif.). Then 100 μl of NaBH₃CN solution (20 mg/ml A. purif.) was added,15 min. at 100° C., then incubated for 16 h at 90° C. The reagents werethen removed against A. purif. or, selectively, by cleansing using aSuperdex S75 column (Pharmacia, Uppsala, Sweden) with 0.1 M TRIS-HClbuffer (pH 7.8) as solvent. The diazotization of the amino groupsintroduced in the SCWPs was done using NaNO₂/HCl following the methoddescribed by Manjon (1985). For the diazotization, 5 mg ofp-phenylenediamine modified SCWP was stirred at 4° C. in 35 μl of of aprecooled mixture of 2 parts NaNO₂ solution (4$ in A. purif.) and 5parts 2 N HCl, and the solution with the diazotized SCWP immediatelybrought into contact with the substances that contained the free aminogroups.

Example 5

Detecting Biological Activity of HF-Extracted, Native and ChemicallyModified SCWP

To detect the specific bonding between the S-layer proteins indicated inthe introduction and the various types of SCWPs, the surface plasmonresonance technique (SPR) was utilized (BIACORE 2000, Biacore, Uppsala,Sweden). The S-layer proteins were immobilized in aqueous form asunassembled monomers and/or oligomers in a concentration of 100 μl/ml A.purif. on pre-activated carboxy-dextran gold chips. The resonance unitsshowed that about 70% of the surface of the chips were coated withS-layer proteins, where the method used resulting in the immobilizationof the sub-units in static alignment. As an example, the studies withthe S-layer protein SbsB of B. stearothermophillus PV72/p2, the S-layerprotein recombinantly made in Escherichia coli (r SbsB₃₂₋₉₂₀;corresponding to pure SbsB of the wild type strain) and an N-terminalshortened form (r SbsB₂₀₆₋₉₂₀; the three SLH motifs are missing) wererepresented. Because of the adsorption curves, it was shown that the

SbsB and r SbsB type B SCWP (concentration range 1–100 μl/ml 50 mMTRIS-HCl buffer, pH 7.2) immobilized on the chips are specificallydetected;

R SbsB₂₅₈₋₉₂₀ (=Δ3 SLH motifs) immobilized on the chips have no affinityto type B SCWP;

SbsB and r SbsB32-920 immobilized on the chips had a comparable affinityfor the type B SCWP that was modified in Example 1–4 as for the nativetype B SCWP;

SbsB and HF-extracted r SbsB32-920 immobilized on the chips do not bindwith lysozyme-digested peptidoglycan.

Comparative tests were also made with the S-layer proteins SbsA, SbsCand r SbS₃₁₋₁₀₉₉ and the type A SCWP, as well as SbpA, r SbpA and thetype C SCWP and SttA and the type D SCWP. In all cases, conclusive proofof the specific bonding between the respective S-layer protein and theassociated SCWP could be demonstrated.

Examples from Group 2: Bonding of Modified SCWPs to Molecules of a SolidSupport for the Directed Monomolecular Bonding of S-Layer Proteins

Example 6

Using Micro-Filtration Membranes (MF) as Solid Support

An MF (Pall carboxydyne) with an average pore size of 0.4 μm and adiameter of 14 mm was used as support. To activate the free carboxylgroups, the MF was placed in a solution of EDC (10 mg/ml A. purif., pH4.7) for 1 h at 20° C., and then washed three times with ice cold A.purif. For the covalent bonding of the type C SCWP, 5 mg of the modifiedmaterial described in Example 1 was dissolved in 5 ml A. purif., wherethe pH value of the solution was set to 9.0 using 0.01 N NaOH. After 4 hof incubation at 20° C., the MF was removed and washed five times with50 mM TRIS-HCl buffer (pH 7.2). Finally, the MF was placed in a solutionof SpbA protein (100 μg/ml 10 nM CaCl₂ solution) and incubated for 6 hat 20° C. To remove the unbound S-layer protein the MF was washed fivetimes with 10 ml 50 mM TRIS-HCl buffer (pH 7.2) and cut into 1 mm² largepieces which were extracted for 10 min at 100° C. with 100 μl of an SDSsolution (sodium dodecylsufate; 10% in A. purif.). Consequently, 25 μlof the clear extract was mixed with 75 μl of the sample solution(Laemmli, 1971), and 1–10 μl of this mixture placed on an SDS gel (10%separation gel). Because of the strength of the protein bands of thesample (molecular weight of SpbA on the SDS gels—127,000) and thecomparison with corresponding SpbA standards, the amount on the boundS-layer protein was estimated at 80–100 μg/cm², which corresponds to themaximum bonding capacity of this highly porous MF for proteins indicatedby the manufacturer.

Example 7

Using a Silicon Oxide Wafer (SOW) as Solid Support

3×5 mm SOW were incubated for 30 min in 500 μl of solution ofaminosilane (5% in A. purif., pH 3.5) at 37° C. After fivefold washingwith A. purif. and ethanol, the silanized SOW were dried for 15 min,then pre-activated for 2 h in a solution ofm-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS, Pierce;150 μg/ml 0.1 M sodium-phosphate buffer, pH 7.0) at 37° C., and withwashed with A. purif. To bind the modified type B SCWP (as described inExample 2), 1 mg of the lyophilisate/ml 0.1 M sodium-phosphate buffer(pH 7.0) was dissolved, and 500 μl applied to the SOW. After 2 h ofincubation at 20° C. in an N₂ atmosphere, the little plates were washedwith 0.1 M sodium-phosphate buffer and A. purif. For the directedbonding either SbsB or r SbsB (100 μg/ml 50 mM TRIS-HCl buffer, pH 7.2)was offered, and the SOW functionalized with type B SCWP incubated for 4h in this solution at 20° C. Washing with TRIS-HCL buffer removed theexcess S-layer proteins, examined the SOW in the AFM and the orientationof the oblique S-layer lattice determined. It was shown that SbSb and rSbsB had bonded with the inner side of the sub-units (N-terminal regionthat carries the 3 SLH motifs). From numerous studies on the SOWs it isknown that SbsB binds with the outer side in the absence of type B SCWP(Neubauer et al, 2000). Consequently, the S-layer sub-units bonded tothe SOW through the type B SCWP covalently bonded to the solid support.Instead of SbsB, recombinant SbSb streptavidin fusion protein can alsobe utilized.

Example 8

Using a Silicon Oxide Wafer (SOW) as Solid Support

For the introduction of thiole groups, a 3×5 mm SOW was placed for 30min in a solution of 3-mercaptosilane (7% in 50% acetone) at 37° C.After fivefold washing with 50% acetone, the SOW was dried for 15 min.at 110° C. in an N₂ atmosphere. To activate thiole groups, 500 μl of asolution of 2–2′ dipyridyldisulfide (5 mg/ml 10% ethanol) was applied tothe SOW. The incubation time was 2 hours at 20° C. After the SOW wasremoved, it was washed several times with 10% ethanol and A. purif. andthen incubated for 6 h in a 500 μl solution of type A SCWP (3 mg/ml A.purif., modified as described in Example 2) at 20° C. To remove surplustype A SCWP, the SOW was washed several times with A. purif., and 50 mMsodium-phosphate buffer (pH 7.0). For the directed bonding, the SOWloaded with type A SCWP was placed in a solution of the followingS-layer protein forms (100 μg/ml 50 mM TRIS-HCl buffer, pH 7.2) for aperiod of 8 h: SbsC (wild type protein), r SbsC₃₁₋₁₀₉₉ and rSbsC₃₄₂₋₁₀₉₉. The N-terminal bonding region responsible for the type ASCWP (Jarosh et al, 2000) was lacking in the last two forms. The SOW waswashed with TRIS-HCL buffer and A. purif. after the incubation with thevarious forms of S-layer protein forms and then examined in the AFM.Because of the oblique S-layer lattice comprising S-layer protein SbsC,a clear determination of the bonding side was possible. It was shownthat the basic vectors had the same orientation as the intact cells orcell wall fragments of B. stearothermophilus ATCC 12980. In the event ofN-terminal shortened r SbsC forms, no lattice structure could bediscovered in the AFM.

Example 9

Using MF as Solid Support

The type A SCWP modified as in Example 1 was bonded to MF activated withcarbodiimide (see Example 6). The following forms of SbsC were offeredin a concentration of 150 μg/ml 50 mM TRIS-HCl buffer (pH 7.2), and themembrane wafers with a diameter of 14 mm incubated for 8 h at 20° C.:SbsC₃₁₋₁₀₉₉, SbsC₂₅₈₋₁₀₀₉, r SbsC₃₄₂₋₁₀₉₉, r SbsC₃₁₋₈₄₄, r SbsC₃₁₋₈₆₀, rSbsC₃₁₋₈₈₀, r SbsC₃₁₋₉₀₀, r SbsC₃₁₋₉₂₀ and SbsC. After the incubationthe MF was washed with 50 mM TRIS-HCl buffer (pH 7.2), cut into 1 mm²sections, extracted with SDS solution and examined with SDS-PAGE (seeExample 6). Because of the respective standard series, the bondedamounts on those forms that carried the complete N-terminus could beestimated between 80–100 μg/cm². On the other hand, no bonding to the MFfunctionalized with type A SCWP took place with r SbsC₂₅₈₋₁₀₉₉ and rSbsC₃₄₂₋₁₀₉₉. The use of various shortened forms confirmed the resultsof the affinity studies (Egelseer et al, 1998; Jarosch et al, 2000) andthe SPR measurements that only the N-terminus is responsible for theinteraction with the SCWP.

Examples from Group 3: Synthesis of Glyco-Lipids (GL) from SCWPs

Example 10

GLs from All Types of SCPWs

The lyophilisated native SCPWS were dissolved in A. purif. (10 mg/ml A.purif.) and mixed with a solution of dipalmitoyl phosphatidylethanolamine (DPPE; 20 mg/40 μl chloroform: methanol=1:1). Afterincubation in screwed reaction tubes at 60° C. for a period of 4 h,NaBH₃CN was added to the solution, and the incubation continued for 16 hat 60° C. The GLs formed were cleaned gel filtration chromatography(Superdex S75). The column eluate was examined for glucosamine andphosphate; every fraction that contained both components were cleanedand lyophilisated.

Example 11

GLs from All Types of SCWPs

Instead of the SCWP with reduced end, the SCPWs modified as in Example 2were used with a free thiole group. DPPE was used as lipid components.To activate the SCWPs, 5 mg were dissolved respectively in 1 ml 50 mMsodium-phosphate buffer (pH 7.0), and 1 mg sulfo-MBS added. Afterincubation for 2 h at 37° C., the reactive mixture was mixed with asolution of DPPE (3 mg in 500 μl chloroform:methanol—1:1=v:v0, and thebatch incubated for 16 h at 20° C. by stirring. In the same manner as inExample 10, the GLs were cleaned using gel filtration chromatographywith the help of a Superdex S75 column.

Examples from Group 4: Using GLs for Making Liposomes and for theDirected Bonding of S-Layer proteins

Example 12

Proof of the Inclusion of GLs in Liposomes

Liposomes which contained DPPC, cholesterine and GL, as described inExample 10 or 11, from type C SCWP at a molecular ratio of 10:5:4 weremade according to the methods described by Mader et al (1999) andcleaned of the output components using gel chromatography. To provewhether the hydrophilic chains of type B SCWP were exposed on thesurface of the liposomes, a periodate oxidation was performed where onlythe vicinal hydroxyl groups were touched. The liposomes that were usedfor the reaction corresponded to a DPPC concentration of 2 μmol/ml 0.2 Msodium-acetate buffer (pH 4.5). As much sodium periodate was added tothis suspension that a final concentration of 20 mM was reached. Thereactive mixture was incubated by stirring for 1 h in the dark at 20° C.After the liposomes were centrifuged and washed twice with 0.2 M sodiumhydrogen carbonate solution (pH 8.2), there were incubated for 4 h in asolution of ferritin (1 mg/ml 0.2 M sodium hydrogen carbonate, ph 8.2)at 20° C. For tests in the transmission electron miscroscope, thesamples were negatively contrasted and embedded for ultra-thin cuts. Thenegatively contrasted preparation showed that the liposome surface wascompletely covered with ferritin; in the ultra-thin cut, a monolayer offerritin could be seen on the liposome surface. After periodateoxidation, the type B SCWP was no longer biologically active; the freealdehyde groups could, however, be used for the covalent bonding of theforeign molecules with the exposed amino groups.

Example 13

Directed Recrystallization of the S-Layer Protein SbsB

The liposomes described in Example 12, containing the GL from DPPE andtype B SCWP (see Example 10) were used for the directedrecrystallization of the S-layer protein SbsB. SbsB bonded with theouter side of liposomes containing DPPE and cholesterol so that theN-terminus is aligned with the bonding regions for the type B SCWP inthe external milieu (Mader et al, 1999). In case of the liposomescontaining the GL from DPPE and type B SCWP, 2 ml of a suspension with atotal DPPE content of 4 μmol was incubated for 6 h with 2 ml of an SbsBsolution (1 mg/ml 50 mM TRIS-HCl buffer, pH 7.2) at 20° C., thesuspension centrifuged to remove surplus S-layer proteins, the liposomeswashed with 50 mM TRIS-HCl buffer (pH 7.2), and the preparation examinedin the transmission electron microscope after freeze-drying at −80° C.and oblique shading with Pt/C. It was shown that in the case of theGL-containing liposomes the smooth outer side of the oblique S-layerlattice was exposed, while with the DPPE-cholesterol liposomes the roughinner side was recognized.

Example 14

Modification of DPPC/cholesterol/HAD-Containing Liposomes with SCWP

Liposomes were made as described by Mader et al (1999). The type C SCWPdescribed in Example 1 and modified with 2-iminothiolan was used tofunctionalize the liposome surface. For this reason, the liposomes thatcorresponded to a DPPC concentration of 5 μmol was suspended in 3 ml 0.2M sodium phosphate buffer (pH 7.0), and 2 mg sulfo-MBS was added to itto activate the free amino groups of HDA. After two hours of incubationat 20° C., it was centrifuged, the liposomes suspended in 2 ml ofsolution of type C SCWP (1.5 mg/ml sodium phosphate buffer, pH 7.0,modified according to Example 2), and incubated for 18 h at 20° C.Proving the immobilization of the SCWP, just like in Example 12, wasdone through periodate oxidation and immobilization of ferritin. Therecrystallization of the S-layer protein, SbpA, was done in a similarmanner was the procedure described in Example 13. The proof of thedirected bonding of the SbpA protein was done using an electronmicroscope on the freeze-dried preparations damped with Pt/C.

Example 15

Using the r SbsB-Streptavidin Fusion Protein

A C-terminal SbsB-streptavidin fusion protein (4 mg lyophilisate)recombinantly prepared in E. coli was dissolved in 2 ml 6 Mguanidinhydrochloride in 50 mM TRIS-HCl buffer (pH 7.2), 0.8 mg ofstreptavidin added and the solution dialyzed for 1 h at 20° C. againstA. purif. After removal of the inner dialysate, it was centrifuged at40,0000×g, and the clear protrusion that contained the soluble rSbsB-strept(4) was placed in a protein concentration of 120 μg/ml 0.2TRIS-HCl buffer (pH 7.2). 1 ml of the protein solution was then mixedwith 1 ml of the liposome suspension that was made as in Example 13, andthe sample incubated for 2 h at 20° C. After the centrifuging, theliposomes were washed twice with 0.2 M TRIS-HCl buffer (pH 7.2), thenincubated with biotinylated human IgC and the bond examined followingthe instructions developed by Mader et al (2000). A bonding ofbiotinylated human IgC was possible only because of the streptavidinresidue exposed on the outside of the S-layer. The desired orientation(bonding through the N-terminus lying over the inside of the S-layer)was observed only for those liposomes where the type B SCWP was bondedcovalently to the HDA.

Examples from Group 5: Layout of Lipid Bilayers

Example 16

Lipid Bilayers with Channel Protein with a Silicon Wafer as SolidSupport

Thiole groups were introduced on one SOW (3×5 mm) through silanizationwith mercaptosilane (see Example 8). Modified type A SCWP (1 mg/ml 50 mMpotassium phosphate buffer, pH 7.0) was bound to these thiole groupsusing sulfo-MBS as a heterobifunctional cross-linker just like inExample 1. After thorough washing of the SOW with phosphate buffer andA. purif., 500 μl of a solution of SbsA protein (100 μg/ml 50 mMTRIS-HCl buffer, pH 7.2) was applied, incubated for 6 h at 20° C.,washed with buffer, and the SbsA protein with glutaraldehyde availableas monolayer (0.5% in 50 mM potassium phosphate buffer, pH 7.2)cross-linked for 20 min at 20° C. A bilyaer of DPPE was then appliedaccording to the method described by Schuster et al (1998), whereα-haemolysin or valinomycin was inserted (Schuster et al, 1998 a, b).

Example 17

Using an S-Layer Ultra-Filtration Membrane (SUM) as Solid Support

An SUM wafer (diameter 25 mm) was placed in a ultra-filtration cell sothat the outer S-layer was exposed. The free carboxyl groups of theS-layer protein were activated for 80 min at 20° C. by the addition ofan EDC solution (6 mg/ml A. purify.; pH 4.7), the membrane surfacewashed three times with ice-cold A. purif., and then 2 ml of a solutionof the type A SCWP modified as in Example 1 (1 mg/ml; pH 9.0) wasapplied for the covalent bonding to the S-layer outer side. After 5 h ofincubation at 20° C., the SUM was washed three times with 50 mM TRIS-HClbuffer (pH 7.2) and three times with A. purif., and then incubated for 5h with a solution of SbsA protein (100 μg/ml 50 mM TRIS-HCl buffer, pH7.2) at 20° C. After the washing of the membrane wafer with buffer, apart was crushed and extracted using SDS solution by means of SDS-PAGEto examine the amount of bonded SbsA. An SbsA-SCWP SUM made in parallelwas cross-linked with glutaraldehyde to stabilize the S-layer protein(as described in Example 16), and used to coat with a DPPE filme andintegral membrane proteins (e.g., α-hameolysin).

Example 18

Covalent Bonding of a GL to an SUM

Using an SUM and coating with the GL from type C SCWP and DPPE describedin Example 10 or 11. Consequently, the SUM functionalized with type CSCWP was incubated for 6 h with a solution of SbpA protein (50 μg/ml mMCaCl₂) at 20° C., and the SUM surface with 10 mM CaCl₂ solution washedthoroughly. To test the directed monomolecular bonding of the SbpAprotein, the SUM was subjected to examinations under the transmissionelectron microscope and freeze-drying, and used for ultra-thin cuts. Itwas shown that a closed monomolecular layer with a cubic lattice waspresent, and the smooth outer side of the S-layer lattice was exposed.

Examples from Group 6: Making SCWP-Containing Copolymers

Example 19

Making a Copolymer by Using Acrylamide

A solution containing 20 mg of type C SCWP modified according to Example1, 6.8 mg acrylamide and 3 μl N,N,N′N′-tetramethylethlenediamine per mlof A. purif. is degassed for 30 min. in a water jet vacuum. After addingammonium peroxydisulfate (1 mg), it is left to react for 18 h at 4° C.Finally, the product is separated using a Sephadex G-50 92.6×100 cm,0.01 M sodium hydrogen carbonate), desalinated using a Biogel P2 andlyophilisated. The SCWP content is determined by amino sugar analysis.

Example 20

Making a Copolymer by Using Polyvinyl Alcohol

A solution of polyvinyl alcohol (PVA; 3% in A. purif.) is made and 20 mgof native type A SCPW are added to 1 ml of this solution. Finally, 100μl are poured out onto a clean container and dried for 30 minutes at 37°C. to stabilize the film. Alternatively, the film can be chemicallysolidified by incubation for 10 minutes in a solution of glutaraldehyde(0.5% in 0.1 M sodium phosphate buffer, pH 7.2). The PVA-SCWP filmwashed with A. purif. is used as a matrix for the directedrecrystallization of the S-layer protein, SbsC (see Examples 8 and 9).

1. A method for constructing a compound body through the use ofbacterial secondary cell wall polymers as bonding agent, said methodcomprising: (a) bringing said polymers into contact with a support insolution, such that a first complex is formed; and then (b) bringingsaid complex into contact with a monomolecular lattice, such that acompound body is formed, comprised of said lattice and said complex. 2.A method according to claim 1, wherein said lattice has a first surfacethat presents secondary cell wall polymer-binding domains.
 3. A methodaccording to claim 2, wherein said lattice has a second surface thatpresents at least one protein comprised of a domain that functionalizessaid compound body.
 4. A method according to claim 1, wherein saidpolymers and said support are covalently linked.
 5. A method accordingto claim 1, wherein said lattice is comprised of crystalline bacterialcell surface layer proteins.
 6. A method according to claim 1, whereinsaid support is a micro-filtration membrane.
 7. A method according toclaim 1, wherein said support is a cell surface layer ultra-filtrationmembrane.
 8. A method according to claim 7, wherein said lattice is alipid layer.
 9. A method for constructing a sandwiched compound bodythrough the use of bacterial secondary cell wall polymers, said methodcomprising: (a) bringing said polymers into contact with crystallinebacterial cell surface layer proteins, such that a monomolecularcrystalline protein layer is formed; (b) bringing said protein layerinto contact with a lipid layer, such that said lipid layer is bound tosaid protein layer through said polymers to form a first aggregatecompound body; (c) repeating steps (a) and (b) to form a secondaggregate compound body; (d) bringing said first aggregate compound bodyinto contact with said second aggregate compound body, such that saidaggregate compound bodies bind to each other through the lipid layers toform a sandwiched compound body.
 10. A method according to claim 9,wherein the protein layer of said first aggregate compound body isattached to a support.
 11. A method according to claim 9, wherein saidfirst and second aggregate compound body are comprised of differentbacterial cell surface layer proteins.
 12. A method according to claim9, wherein said support is a micro-filtration membrane.
 13. A methodaccording to claim 9, wherein said support is a cell surface layerultra-filtration membrane.
 14. A method according to claim 8, whereinsaid monomolecular lattice is in the form of a liposome.
 15. A methodaccording to claim 1, wherein said support is a micelle comprised ofhydrophobic self-assembling chains.